Naturalkillercells(NK)actasafirstlineofdefenseagainstinfectionandcancer.NKactivationiscontrolledby a balance of signals transmitted by activatingand inhibitory NK receptors. The activating receptor NKp46 is considered the major activating receptor in natural cytotoxicity against autologous, allogeneic and xenogeneic target cells. NKp46 is conserved in mammals and virtually ubiquitously expressed on NK, suggesting it is functionally important. Mice deficient in Ncr1, the gene encoding NKp46, are impaired in tumor immune surveillance,havemoresevereinfluenzaA,metapneumovirus,reovirusandfusobacteriuminfectionsandgraft versus host disease. The endogenous ligand of this important activating NK receptor is not known, despite decades of searching. Our preliminary work suggests that NKp46 recognizes calreticulin (CRT), which is normallyfoundinsidetheendoplasmicreticulum(ER)butgetstransferredtothecellsurfaceinER-stressedcells or tumor cells treated with some cancer drugs.An NKp46-Ig fusionprotein specifically pulls downcell-surface CRT (ecto-CRT) and preliminary data using surface plasmon resonance indicate specific binding. Knocking down CALR, the gene encoding for CRT, or adding blocking antibodies to CRT inhibits NKp46-mediated NK killing.Somechemotherapeuticagentsandradiotherapyinduceanimmunostimulatorytypeofprogrammedcell death in cancer cells, known as immunogenic cell death (ICD), which removes surviving tumor cells after treatment. The mechanismbehind ICDhasbeenlinked toecto-CRT, which serves as an ?eat me?/phagocytic signalfordendriticcells,whichthenactivateanti-tumorCD8+Tcellsbycross-priming.Herewehypothesizethat NKp46recognizesecto-CRT,andthatNKrecognitionofER-stressedcellsviaNKp46interactionwithecto-CRT playsanimportantroleinICDandNK-mediatedimmunedefensemoregenerally.Inpreliminarydata,infection withZIKV,whichreplicatesintheERandcausesERstress,andtreatmentoftumor celllineswithICD-inducing chemotherapy drugs induces ecto-CRT and NKp46-dependent NK killing. Moreover, CRT-coated tumor cells becomeNKcelltargets,andNCR1knockoutinanNKcelllinestronglyinhibitsitskillingoftargetswithexposed CRT. To test our hypotheses, we will first confirm that ecto-CRT is a ligand for NKp46 by using biophysical methods to characterize ecto-CRT and NKp46 binding and identifying the regions of CRT responsible for the interaction.Genetic manipulationor blockingof NKp46on NKandof ecto-CRT ontumortargets will examine theroleofthisreceptor-ligandinteractioninNKrecognition,immunesynapseformationandfunctionalresponses to infected and tumor cells. NK killing of untreated tumor cells and tumor cells treated with ICD-inducing and noninducingdrugswillbecompared.TheinvivoroleofNKp46incontrollingZIKVinfectionandmousetumors (withoutandwithchemotherapy)willbeassessedbycomparingvirallevelsandtumorgrowth,NKcellinfiltration oftumorsandsurvivalinwild-typeandNcr1-/-mice.